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Pseudomonas aeruginosa is a pathogen of critical priority importance according to the WHO. Due to its multi-resistance and expression of various virulence factors, it is the causal agent of severe healthcare-acquired infections (HAIs). Effective strategies to control infections caused by P. aeruginosa must include early and specific detection of the pathogen for early and timely antibiotic prescription. The need to develop a specific and reproducible diagnostic technique is urgent, which must often be more sensitive and faster than current clinical diagnostic methods. In this study, we implement and standardize the loop-mediated isothermal amplification (LAMP) technique, coupled with the reverse line blot hybridization (RLBH) technique for the detection of P. aeruginosa. A set of primers and probes was designed to amplify a specific region of the P. aeruginosa 16s rRNA gene. The sensitivity of the LAMP-RLBH method was 3 × 10−4 ng/μL, 1000 times more sensitive than the PCR and LAMP technique (this work), with a sensitivity of 3 × 10−3 ng/μL. The LAMP-RLBH and LAMP techniques showed specific amplification and no cross-reaction with members of the ESKAPE group and other Pseudomonas species. The present investigation provides a technique that can be easily performed in less time, achieving a faster and more reliable alternative compared to traditional microbial diagnostic methods for the detection of P. aeruginosa. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a pathogen of critical priority importance according to the WHO. Due to its multi-resistance and expression of various virulence factors, it is the causal agent of severe healthcare-acquired infections (HAIs). Effective strategies to control infections caused by P. aeruginosa must include early and specific detection of the pathogen for early and timely antibiotic prescription. The need to develop a specific and reproducible diagnostic technique is urgent, which must often be more sensitive and faster than current clinical diagnostic methods. In this study, we implement and standardize the loop-mediated isothermal amplification (LAMP) technique, coupled with the reverse line blot hybridization (RLBH) technique for the detection of P. aeruginosa. A set of primers and probes was designed to amplify a specific region of the P. aeruginosa 16s rRNA gene. The sensitivity of the LAMP-RLBH method was 3 × 10−4 ng/μL, 1000 times more sensitive than the PCR and LAMP technique (this work), with a sensitivity of 3 × 10−3 ng/μL. The LAMP-RLBH and LAMP techniques showed specific amplification and no cross-reaction with members of the ESKAPE group and other Pseudomonas species. The present investigation provides a technique that can be easily performed in less time, achieving a faster and more reliable alternative compared to traditional microbial diagnostic methods for the detection of P. aeruginosa.

Persistent HPV infection is a major risk factor for the subsequent development of cervical cancer. LAMP is simple and suitable for field detection in the resource-limited settings. In this study, hydroxy naphthol blue (HNB)-based visual LAMP and evagreen-based fluorescent LAMP coupled with a microfluidic chip (LAMP-chip) were established for the field detection of seven subtypes of HPV. The analytical sensitivity was 19–233 copies/reaction. The overall clinical sensitivity was 97.35% for visual LAMP and 98.23% for LAMP-chip. Both LAMP assays exhibited 100% specificity and were completed in less than 50 min. Additionally, both assays did not require complicated nucleic acid extraction and purification steps. A complete quality control monitoring system (including internal control, positive quality control and negative control) in the LAMP assays further ensured the credibility of the results. Our findings demonstrated that the proposed LAMP assays have the potential to be applied in the testing of common HPV DNA in field investigations (visual LAMP) or within communities and primary health centers (LAMP-chip).

Persistent HPV infection is a major risk factor for the subsequent development of cervical cancer. LAMP is simple and suitable for field detection in the resource-limited settings. In this study, hydroxy naphthol blue (HNB)-based visual LAMP and evagreen-based fluorescent LAMP coupled with a microfluidic chip (LAMP-chip) were established for the field detection of seven subtypes of HPV. The analytical sensitivity was 19-233 copies/reaction. The overall clinical sensitivity was 97.35% for visual LAMP and 98.23% for LAMP-chip. Both LAMP assays exhibited 100% specificity and were completed in less than 50 min. Additionally, both assays did not require complicated nucleic acid extraction and purification steps. A complete quality control monitoring system (including internal control, positive quality control and negative control) in the LAMP assays further ensured the credibility of the results. Our findings demonstrated that the proposed LAMP assays have the potential to be applied in the testing of common HPV DNA in field investigations (visual LAMP) or within communities and primary health centers (LAMP-chip).

Abstract Whooping cough also called Pertussis is a highly contagious respiratory infection that affects all age populations. Given recent pertussis outbreaks, there is an urgent need for a point-of-care (POC) device for rapid diagnosis of pertussis. Herein, we report a low-cost microfluidic POC device integrated with loop-mediated isothermal amplification (LAMP) technique for the rapid and accurate diagnosis of pertussis. The 3D-printed bioanalyzer housed not only the biochip but also an in-house-developed portable and fully battery-powered heater for rapid POC detection of pertussis, without the need of external electricity. The fluorescence-based results could be rapidly visualized in about one hour by the naked eye without the need for any additional instrumentation. In addition, a simple centrifuge-free sample preparation process was optimized for the efficient lysis of pertussis samples and successfully used for direct detection of bacteria in nasopharyngeal samples. High sensitivity, with a limit of detection (LOD) of 5 DNA copies per LAMP zone, and high specificity were demonstrated. We envision that the microfluidic POC device can be used in various venues such as medical clinics, schools, and other low-resource settings for the fast detection of pertussis. Graphical abstract We report a low-cost microfluidic point-of-care (POC) platform integrated with loop-mediated isothermal amplification (LAMP) supported by a portable battery-powered heater for rapid and instrument-free detection of whooping cough in resource-limited settings. Image 1 Highlights • We for the first time report a low-cost hybrid microfluidic platform integrated with LAMP for rapid and instrument-free detection of whooping cough (pertussis). • We developed a portable and fully battery-powered heater to support on-chip LAMP reactions, which significantly enhanced the capacity for point-of-care detection without the requirement of the external AC power supply. • The microfluidic approach demonstrated high specificity by detecting B. pertussis and other Bordetella species. The limit of detection of 5 DNA copies per LAMP zone was achieved, demonstrating high detection sensitivity. • By using an optimized centrifuge-free lysis protocol, direct detection of B. pertussis bacteria was achieved. • Our novel microfluidic approach has great potential for point-of-care detection of many other pathogens such as foodborne pathogens, especially for low-resource settings. [ABSTRACT FROM AUTHOR]

In classical biocontrol programmes, a rapid and correct identification of the introduced antagonist is a key issue during both the release and establishment monitoring phases. It is often difficult to distinguish morphologically cryptic species or immature stages. An accurate diagnosis can now be provided by molecular diagnostic methods. Among the conventional and real-time PCR-based methods, loop-mediated isothermal amplification (LAMP) is a particularly suitable technique as it allows a rapid amplification of target DNA directly in the field. During the programme implemented in Italy against the Asian chestnut gall wasp (ACGW)Dryocosmus kuriphilus, we developed a real-time LAMP assay, combined with a simple DNA extraction method, for rapid in-field identification of larvae, pupae, and adults of the biocontrol agent, the parasitoidTorymus sinensis. Validation of the assay comprised adults as well as preimaginal stages of parasitoids obtained from ACGW galls collected from different localities. Results confirmed the effectiveness of the LAMP assay to rapidly and specifically identify the target parasitoid in the field. This assay will be a valuable tool for quick on-site checking of the parasitism rate. [ABSTRACT FROM AUTHOR]